Human gammaherpesviruses KSHV and EBV establish long-term latent infections causally associated with human malignancy in AIDS patients. The establishment, maintenance and reactivation of the latent viral genome are processes essential for virus survival and pathogenesis, and represent potential targets for antiviral therapies. During latency, the viral genomes persist as multicopy episomes packaged with cellular chromatin-associated proteins. We hypothesize that chromatin-related factors contribute significantly to the stable maintenance of the latent viral genome and organize gene expression patterns that enforce latent gene transcription, and limit lytic cycle reactivation. In this application, we propose to investigate the role of chromatin organization and modification in the regulation of three critical regulatory regions of the KSHV genome. In particular, we will investigate the role of repressive chromatin at the immediate early gene ORF50 (Rta) promoter that restricts lytic cycle activation. We will also investigate the chromatin structure and cell cycle changes in chromatin at the KSHV origin of plasmid replication found in the terminal repeats (TR). Finally, we will investigate chromatin organization of the major latency promoter regulating the multicistronic transcript for ORF72 (LANA), ORF73 (vCyclin), and K13 (vFLIP). The molecular biology of KSHV gene expression, chromatin organization, and genome maintenance during latency may have significant impact on our understanding of KSHV pathogenesis in AIDS associated lymphomas. It is particularly valuable to understand the mechanism of episomal stability, which underlies that ability of KSHV genomes to persist in latency. It is also valuable to understand the mechanism of latent cycle transcription of the ORF72-ORF73-K13, which may contribute directly to KSHV-associated neoplasia. These studies are also likely to provide important insight into the shared properties of other herpesviruses, especially EBV, which may utilize similar strategies for genome stability during latency in B-lymphocytes. Finally, these studies may provide novel insights into basic mechanisms of chromosomal stability and organization, which may underlie chromosomal instability defects found in many pre-cancerous lesions. [unreadable] [unreadable]